This invention relates to a system for the liquid chromatography as a separating or analysing instrument, commonly used in the chemical, biological, medical, and the like science fields, and a liquid supplying method employed for supplying an eluent for the abovementioned purpose. More particularly it relates to an eluent preparing and pumping system for use in a so-called high speed chromatography apparatus; in other words, to a provision of a novel pumping system, enabling a non-pulsating sucking of liquids and perfect mixing thereof as the most important factors, and an eluent supplying method for that purpose.
More specifically still, it relates to a method and system for producing an eluent having a time varying concentration of different liquids.
In general, the simplest elution system, used for example in a liquid-chromatographic column, supplies an eluent comprising a single liquid to the column. In many instances, however, this single liquid is not effective in removing all of the desired material from the column, and a second liquid must subsequently be supplied. Alternately, an eluent having a set concentration of the two liquids is used. More than two liquids can be and have been used, but for convenience in describing the prior art and the present invention, the discussion is limited in what follows to a system using two liquids which are labelled as liquid A and liquid B, in general. Switching liquids in mid-operation and the use of a mixture of liquids have not been favored procedurally, and there has been a recent tendency to gradient elution systems. In gradient elution, the initial eluent contains a set concentration of the liquids (usually 100% of A and none of B), and this concentration is slowly altered, through intermediate concentration, to a second set concentration of the liquids (usually none of A and 100% of B).
Required conditions for the pumping system in a liquid chromatography used in such a gradient elution system are: (1) mixing of liquids at a precise and freely variable concentration with a gradual and stepless gradient: (2) constant flow rate especially in an almost non-pulsating state, referred to hereafter as low-pulsation; (3) resistance against high pressure, that is, capacity of attaining a considerably high flowing rate even inside a small-diametered column, etc.
Conventional apparatuses, as one of the representative example thereof FIG. 1 being shown, have been in an unsatisfactory state from the abovementioned viewpoint. In this apparatus, each liquid A and B filled in each independent liquid source container (hereinafter simply called liquid container) 1 and 2 is sucked into, by means of each pump 3 and 4, each suction pipe or tube 5 and 6, and then sent into a mixing-and-stirring chamber (hereinafter simply called mixing chamber) through each discharge pipe 7 and 8, and finally led, through a liquid supply pipe 10, a sample injecting pipe 11, and a column 12, into a detector 13. The sample is timely or at a suitable interval injected, at a sample injecting pipe 11 disposed on the way of the liquid supply pipe 10 by means of a sample injector 14. Both liquids A and B are respectively flow-controlled by a flow-rate programmer 15, at a set flow rate, into a mixture of desired concentration, and further formed into an eluent with a time varying concentration. And the pair of pumps 3 and 4 are varied, in the course of the above operation, respectively in the number of rotation for varying the ratio of discharge amount of each liquid, thereby timewise varying the ratio of the discharge amount.
This type method and apparatus therefor is, however, still not free from some disadvantages: one pump is required for each liquid; the pump used therein cannot be said to be a low-pulsation pump; viscosity, compressibility, vapor pressure, etc., peculiar to each liquid tend to hinder a precise proportioning of the number of pump rotations to the mixing ratio of the liquids. Difficulty of preparing an eluent having a precise mixing ratio of A and B liquids in this programming system may be said to be a decisive disadvantage to a gradient elution system which essentially requires a precise controlling of the liquids concentration.
Furthermore, another elution system was made public in Toku-Ko-Sho No. 50-19959 in Japan as follows:
An elution system comprising:
(a) a source of first liquid; PA1 (b) a source of second liquid; PA1 (c) first liquid supply means connected to said source of first liquid; PA1 (d) second liquid supply means connected to said source of second liquid; PA1 (e) proportioning means comprising a mixing region, valving means connected to said mixing region, and programming means connected to said valving means, said valving means having at least two operative portions, a first operative portion connected between said first liquid supply means and said mixing region in a manner such as to supply said mixing region with said first liquid when said first operative portion is activated and a second liquid supply means and said mixing region in a manner such as to supply said mixing region with said second liquid when said second operative portion is activated, said programming means being adapted to periodically control the time during which each operative portion of said valving means is activated, said mixing region comprising means to allow a portion of any liquid contained in said mixing region to be removed from said mixing region.
Some disadvantages are still to be pointed out even in this elution system, most of those coming from a the fact that each liquid is obliged to be mixed under a high pressure, because the pump is disposed between each liquid container and each valving means for supplying the liquid to the valving means thereby. The mixing of liquids under this high pressure still provides several problems involved therein. In this system, controlling of the liquid supply is carried out by the valving means instead of the conventional method of doing it by the number of pump rotations, which allows a fairly precise mixing ratio between each liquid, to be sure, but still is not free from some deviation from the predetermined program value of the mixing ratio, because of the mixing process under the high pressure. Difference of compressibility of each liquid may be a main cause of the abovementioned deviation, when the mixing is carried out under the condition of high pressure like this. Although the compressibility of liquids is smaller than that of gases, even a slight difference between liquids of this inherently small liquid compressibility can affect the mixing ratio of liquids in the present day precise liquid chromatography which requires a high speed analysis, by flowing the eluent at a high speed into the column, under a high pressure more than 50 Kg/cm.sup.2 or sometimes over 100 Kg/cm.sup.2. A minute variation of the mixing ratio of the liquids derived from the difference of the compressibility between liquids can cause an innegligible increasing of analysis errors.
Occurrence of pulsating flow of liquid, which is observed on the outlet or discharge side by dint of a pump disposed in this type of elution system, renders the base line of a chromatogram described in the liquid chromatography unstable. This also provides another problem giving rise to an imprecise or inaccurate quantative analysis.